Ana Sampaio

Enzymatic saccharification of biologically pre-treated wheat straw with white-rot fungi

Wheat straw was submitted to a pre-treatment by the basidiomycetous fungi Euc-1 and Irpex lacteus, aiming to improve the accessibility of cellulose towards enzymatic hydrolysis via previous selective bio-delignification. This allowed the increase of substrate saccharification nearly four and three times while applying the basidiomycetes Euc-1 and I. lacteus, respectively. The cellulose/lignin ratio increased from 2.7 in the untreated wheat straw to 5.9 and 4.6 after the bio-treatment by the basidiomycetes Euc-1 and I. lacteus, respectively, thus evidencing the highly selective lignin biodegradation. The enzymatic profile of both fungi upon bio-treatment of wheat straw have been assessed including laccase, manganese-dependent peroxidase, lignin peroxidase, carboxymethylcellulase, xylanase, avicelase and feruloyl esterase activities. The difference in efficiency and selectivity of delignification within the two fungi treatments was interpreted in terms of specific lignolytic enzyme profiles and moderate xylanase and cellulolytic activities.

Environmental distribution of Cryptococcus neoformans and C. gattii around the Mediterranean basin

In order to elucidate the distribution of Cryptococcus neoformans and C. gattii in the Mediterranean basin, an extensive environmental survey was carried out during 2012-2015. A total of 302 sites located in 12 countries were sampled, 6436 samples from 3765 trees were collected and 5% of trees were found to be colonized by cryptococcal yeasts. Cryptococcus neoformans was isolated from 177 trees and C. gattii from 13. Cryptococcus neoformans colonized 27% of Ceratonia, 10% of Olea, Platanus and Prunus trees and a lower percentage of other tree genera. The 13 C. gattii isolates were collected from five Eucalyptus, four Ceratonia, two Pinus and two Olea trees. Cryptococcus neoformans was distributed all around the Mediterranean basin, whereas C. gattii was isolated in Greece, Southern Italy and Spain, in agreement with previous findings from both clinical and environmental sources. Among C. neoformans isolates, VNI was the prevalent molecular type but VNII, VNIV and VNIII hybrid strains were also isolated. With the exception of a single VGIV isolate, all C. gattii isolates were VGI. The results confirmed the presence of both Cryptococcus species in the Mediterranean environment, and showed that both carob and olive trees represent an important niche for these yeasts.

Influence of ligninolytic enzymes on straw saccharification during fungal pretreatment.

Solid state and submerged fermentations in the presence of white-rot basidiomycetes (Bjerkandera adusta, Fomes fomentarius, Ganoderma resinaceum, Irpex lacteus, Phanerochaete chrysosporium, Trametes versicolor and basidiomycete Euc-1) and the litter-decomposing basidiomycete Lepista nuda were evaluated as a pretreatment to increase enzymatic saccharification of wheat straw. Enzymatic hydrolysis of holocellulose after solid state pretreatment showed a significant (P<0.05) increase of saccharification process for T. versicolor, Euc-1, G. resinaceum and I. lacteus, being T. versicolor (strain Tv2) the best one with a sugar yield increase of 91% compared with untreated straw. In submerged medium the pretreatment with I. lacteus, Euc-1 and P. chrysosporium enhanced saccharification but at a lesser extent. Covariance analysis was used to evaluate the relationships between ligninolytic enzymes (lignin peroxidase, manganese-dependent peroxidase and laccase) and saccharification increase. Results showed that only the presence of lignin peroxidase during pretreatment can lead to a significant (P<0.05) increase in the saccharification yield.

Invertebrate and microbial colonisation in native and exotic leaf litter species in a mountain stream

Decomposition of three leaf species (Alnus glutinosa, Eucalyptus globulus and Quercus robur) were examined in a headstream. During two months decomposing leaves were periodically analysed for nutrient content, soluble sugars, phenols, protein precipitation capacity, total fiber, weight loss, microbial and macroinvertebrate colonisation. The leaves of the three species showed similar patterns in dynamics of soluble sugars, tannins and phenols. Bacteria numbers per foliar dry mass were constant in oak during the experiment, but increased linearly in eucalyptus. Total heterotrophic colony forming units (bacteria and fungi) were similar in eucalyptus and oak and constant during incubation, whereas in alder, they became more abundant. The analysis of invertebrate assemblages revealed differences between alder and the other two species related to nitrogen and microbial abundance.

Importance of Resolving Fungal Nomenclature: the Case of Multiple Pathogenic Species in the Cryptococcus Genus

Cryptococcosis is a major fungal disease caused by members of the Cryptococcus gattii and Cryptococcus neoformans species complexes. After more than 15 years of molecular genetic and phenotypic studies and much debate, a proposal for a taxonomic revision was made. ABSTRACT Cryptococcosis is a major fungal disease caused by members of the Cryptococcus gattii and Cryptococcus neoformans species complexes. After more than 15 years of molecular genetic and phenotypic studies and much debate, a proposal for a taxonomic revision was made. The two varieties within C. neoformans were raised to species level, and the same was done for five genotypes within C. gattii. In a recent perspective (K. J. Kwon-Chung et al., mSphere 2:e00357-16, 2017, https://doi.org/10.1128/mSphere.00357-16 ), it was argued that this taxonomic proposal was premature and without consensus in the community. Although the authors of the perspective recognized the existence of genetic diversity, they preferred the use of the informal nomenclature “C. neoformans species complex” and “C. gattii species complex.” Here we highlight the advantage of recognizing these seven species, as ignoring these species will impede deciphering further biologically and clinically relevant differences between them, which may in turn delay future clinical advances.

Winery wastewater treatment by combination of Cryptococcus laurentii and Fenton's reagent.

Winery wastewaters (WW) have high levels of organic matter, resulting in high COD and BOD and suspended solids. This paper studies the combination of biological and chemical processes in WW treatment. Among 10 yeast isolates, Filobasidium sp. (AGG 577) and Cryptococcus laurentii (AGG 726) were selected due to their superior performance in COD removal. During WW degradation, COD and total polyphenols (TPP) content removal of 89-90% for Filobasidium sp. and 90-93% for C. laurentii were obtained. However, despite similar degradation efficiency for both yeasts, COD kinetics and pH evolution during treatment reveals that C. laurentii presents a faster response than Filobasidium sp. The toxicity (inhibition of Vibrio fischeri luminescence) of C. laurentii treated WW decreases to an inhibition value below 2.5%. However, treated WW exceeds the legal limits, making necessary an additional treatment. In this case, the selection of Fentons reagent as a chemical final polish step process is a good compromise between efficiency and lower practical complexity. The best results for both COD and TPP removal were obtained with H2O2 initial concentration of 39.2mM and a H2O2:Fe(2+) molar ratio of 15:1. The combined C. laurentii - Fentons reagent treatment of WW achieved a total reduction of 98% and 96%, for COD and TPP, respectively.

Decolorization of Azo Dyes by Yeasts

Azo dyes are synthetically produced organic molecules and represent the largest group of commercial dyes. Industrial use for coloring purposes generates huge volumes of dyed effluents, which are of environmental concern. Color removal has been achieved by using microorganisms such as filamentous fungi, especially white rot fungi, and bacterial species. In this chapter, we look for a still largely unexplored microbial group – the yeasts, and based on the review of current state of the art, we discuss the potential biotechnological applications in the field of azo dyes bioremediation.

Direct and specific identification of Cryptococcus neoformans in biological samples using fluorescently labelled DNA probes

Fluorescence in situ hybridisation (FISH) is a suitable technique for the rapid, reliable and cultivation-independent identification of microbial pathogens. This study describes the development of fluorescently labelled rRNA-targeted oligonucleotides and a FISH assay to detect and identify Cryptococcus neoformans in culture and biological samples. All C. neoformans reference and clinical isolates gave positive signals with the specific oligonucleotide probes, whereas all non-target yeast species gave negative reactions with the same probes. The assay was also successfully applied to the detection of C. neoformans cells in cerebrospinal samples from patients with clinical diagnosis of cryptococcosis. The described FISH-based assay revealed to be practical, sensitive and specific for the detection and identification of C. neoformans yeasts.

Genotypic diversity of environmental Cryptococcus neoformans isolates from Northern Portugal

The Cryptococcus neoformans/C. gattii species complex members are the main agents of systemic cryptococcosis. This disease is believed to be acquired from the environment via fungal cell inhalation. Often, isolates recovered from environmental and clinical sources have proven to be genotypically similar. We assessed the occurrence of C. neoformans and C. gattii in environmental substrates collected in a Portuguese region. Twenty‐eight isolates were identified as C. neoformans – five from decaying Eucalyptus leaves and 23 from domestic pigeon droppings. The isolates were genotyped using a URA5‐RFLP approach. The C. neoformans VNIV (53.6%, n = 15) and VNI (32.1%, n = 9) genotypes were abundantly present among environmental isolates. The hybrid VNIII (14.3%, n = 4) genotype was underrepresented and the VNII was not found. Cryptococcus gattii was also not found although some isolates yielded a positive canavanine–glycine–bromothymol blue test.

Environmental Applications of Fungal and Plant Systems: Decolourisation of Textile Wastewater and Related Dyestuffs

Wastewater treatment plants, such as activated sludge and methanogenic reactors, are not the natural habitat of WRF, since these organisms prefer solid substrates and well-aerated environments. The fact, that constructed wetlands (e.g. sub-surface flow systems with rooted emergent macrophytes), are transitional environments, i.e. are intermediate between terrestrial and aquatic ecosystems, can be an advantage in the treatment of polluted effluents. The wetlands system treats wastewater by physical, chemical and biotic processes, in a close association of appropriated plants, microorganisms, macro-organisms and substrates. Macrophytes enhance physical filtration, prevent clogging in vertical flow systems, mediate oxygen transfer to the rhizosphere and favour microorganism colonisation (Brix et al. 1996; Brix 1997). In sub-surface systems, there is an oxygen gradient, with high partial pressures near the plant roots, to be replaced progressively by anaerobic and anoxic environments. The mixture of aerobic, anoxic and anaerobic zones stimulates different microbial communities that can degrade complex organic substances (such as azo dyes) almost to mineralisation. The extent of dyes biodegradation must be evaluated, since the formation of intermediate compounds can enhance toxicity (Sweeney et al. 1994). The use of constructed wetlands is a low cost technique, with low maintenance needs (Schwitzguebel et al. 2002; Susarla et al. 2002). It is able to tolerate high fluctuations in flow, temperature (Winthrop et al. 2002) and the composition and/or concentration of pollutants in wastewater. Finally, it is likely to find widespread acceptance with the public for its obvious technological and aesthetic qualities.